Lectin-derived progenitor cell preservation factors and methods of use

ABSTRACT

The invention relates to an isolated nucleic acid molecule that encodes a protein that is effective to preserve progenitor cells, such as hematopoietic progenitor cells. The nucleic acid comprises a sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof. The encoded protein has an amino acid sequence that comprises a sequence defined by SEQ ID NO:2, a homolog thereof, or a fragment thereof that contains an amino acid sequence TNNVLQVT. Methods of using the encoded protein for preserving progenitor cells in vitro, ex vivo, and in vivo are also described. The invention, therefore, include methods such as myeloablation therapies for cancer treatment wherein myeloid reconstitution is facilitated by means of the specified protein. Other therapeutic utilities are also enabled through the invention, for example, expanding progenitor cell populations ex vivo to increase chances of engraftation, improving conditions for transporting and storing progenitor cells, and facilitating gene therapy to treat and cure a broad range of life-threatening hematologic diseases.

BACKGROUND OF THE INVENTION

The invention relates to a nucleic acid and its corresponding proteinfor use in connection with the preservation of progenitor cells. Morespecifically, the invention relates to a nucleic acid and the proteinthat it encodes and which is capable of preserving progenitor cells, aswell as a method of using the protein for preserving progenitor cells.

Each day the bone marrow generates and releases into the circulationseveral billion fully-differentiated, functional blood cells. Productionof these cells derives from a small stock of quiescent progenitor cells(including the most primitive stem cells and other less primitive butstill immature progenitors) by a process called hematopoiesis (Zipori1992). The most primitive stem cells have the capacity to generate >10¹³cells containing all blood lineages (Turhan et al. 1989). The productionof such a large number of cells is achieved by extensive proliferationcoupled with successive differentiation steps leading to a balancedproduction of mature cells. Progenitor cells progressively lose theircapacity to generate multiple cells lineages and eventually producecells of one or two cell lineages.

Soluble regulators and cell-cell interactions mediate differentiationpathways of immature progenitors through a tightly-controlled butinadequately understood process. Several of the body's soluble factorshave been isolated and characterized both in culture and in animals(see, e.g., Ogawa (1993) and references therein). Regulators such as thecolony stimulating factors (e.g., IL3, GM-CSF, G-CSF, M-CSF) not onlyinduce proliferation and differentiation of progenitors capable ofproducing cells of either multiple cell lineages (IL3 and GM-CSF) orsingle cell lineages (G-CSF and M-CSF), but also preserve viability oftheir respective progenitors for short periods. Other regulators such asinterleukin-1 (IL1), the kit ligand (KL), and thrombopoietin (Borge etal. 1996) increase viability of multipotential progenitors in additionto other functions. No known cytokines alone or in combination canpreserve viability of primitive progenitors in liquid culture withoutstromal support beyond a few days.

Regulation of primitive stem cells appears to differ from that ofimmature, multilineage progenitors. Hematopoietic stem cells, whichreside in the bone marrow predominantly in a quiescent state, do notappear to respond immediately to regulators that induce differentiationand proliferation. Maintenance of these cells in the body is mediatedvia cell-cell interactions and soluble regulators. Maintenance ofquiescent stem cells in vitro has been achieved by culturing cells onadherent stromal layers with soluble regulators such as IL3, IL6, KL andLIF (Young et al. 1996). Recently, the addition of the FLK2/FLT3 ligand(FL) to this complex culture has been found to extend maintenance ofquiescent stem cells from a few weeks to three months (Shah et al.1996).

While the use of stromal cell culture has heretofore proven to be usefulfor the maintenance of hematopoietic stems cells in the laboratorysetting, such approaches are not easily amenable to clinicalapplication. Isolating and establishing stromal cell cultures forindividual patients is not practical either because of time constraintsor because a patient's marrow may be compromised by the underlyingdisease or exposure to agents (e.g., radiation, chemotherapy) that candamage the bone marrow microenvironment.

Lectins, defined as carbohydrate-binding proteins other than antibodiesor enzymes, (Baronedes 1988), are widespread among plants, prokaryotes,and eukaryotes (see generally, Gabius et al. 1993). Each lectinrecognizes a specific carbohydrate moiety, and forms a non-covalent bondwith the carbohydrate through a stereochemical fit of complementarydomains (e.g., hydrophobic pocket). Carbohydrates are widely present oncell surfaces (in the forms of glycoproteins, glycolipids, andpolysaccharides), and appear to mediate cell-cell contacts includingcell recognition (Sharon et al. 1989). Abnormal glycosylation patternsare associated with disease by causing alterations in a protein'ssurface properties, conformation, stability, or protease resistance(Dwek 1995).

Gowda et al. (1994) described the isolation of amannose-glucose-specific lectin from the hyacinth bean (Dolichos lablab). Purification and sequencing of this lectin is said to indicatethat the protein includes two nonidentical subunits. The Gowda et al.publication describes evolutionary relationships of the lectin to otherlectins, but does not ascribe any function to the protein beyondsaccharide-binding.

Cell agglutinating properties of certain plant lectins have been knownfor over 100 years. Certain lectins have been used as tools inimmunology laboratories as potent, specific activators of T lymphocytes(phytohemagglutinin (PHA) and concanavalin A (ConA)) and B lymphocytes(pokeweed mitogen (PWM)) for over 30 years (Sharon et al. 1989). Somelectins have also been used to isolate hematopoietic progenitors forover 15 years (Gabius 1994a). Large numbers of cancer patients in Europehave received crude extracts of mistletoe lectin (Viscum album)intravenously as a candidate cancer therapy without major complications(Gabius 1994b). Whether these plant lectins act on mammalian cells viade novo means, or simply mimic their functional mammalian homologs isnot yet known. No lectin has yet been successfully developed as a humantherapeutic.

In view of the above considerations, it is clear that regulation of thehematopoietic process remains incompletely understood. Most solubleregulators identified, such as the colony stimulating factors andinterleukins, induce proliferation and differentiation of progenitorscells in culture and their levels in the blood circulation increaseduring times of hematopoietic stress (e.g., blood loss, infection). Forexample, U.S. Pat. No. 4,808,611 describes a method of using IL1 and acolony stimulating factor to induce proliferation and differentiation ofhemopoietic stem cells. Some soluble regulators, such as IL1, IL6, IL11,KL, FL, and Tpo, marginally increase viability of primitive progenitorson their own, but when added in combination induce proliferation anddifferentiation of progenitors. Soluble regulators that maintain orexpand primitive progenitors for extended periods in the absence ofstromal support are not yet commercially available. As a consequence,numerous potential therapeutic approaches to diseases such as cancer andgenetic blood diseases remain unexplored.

Accordingly, it is one of the purposes of this invention to overcome theabove limitations in methods of regulating hematopoietic processes, byproviding a factor and method of protecting, preserving, and expandinghematopoietic progenitor cell populations. It is another purpose of theinvention to provide means for protecting the integrity of thehematopoietic processes in vivo as an adjunct to therapeutic treatmentsrelated to cancer and other diseases that can otherwise adversely impactupon the hematopoietic system.

SUMMARY OF THE INVENTION

It has now been discovered that these and other objectives can beachieved by the present invention, which provides an isolated nucleicacid comprising a nucleotide sequence as defined by SEQ ID NO:1, ahomolog thereof, or a unique fragment thereof that encodes an amino acidsequence TNNVLQVT.

The isolated nucleic acid preferably encodes a mannose/glucose-specificlegume lectin, and is more preferably isolated from a legume of thetribe Phaseoleae. Most preferably, the protein is encoded by a nucleicacid that is isolated from red kidney beans, white kidney beans,hyacinth beans, or black-eyed peas. The isolated nucleic acid of theinvention preferably comprises a nucleotide sequence as defined by SEQID NO:1 or a unique fragment thereof.

Also, the protein encoded by the nucleic acid of the invention iscapable of preserving progenitor cells that are at least unipotentprogenitor cells, but the protein can be used to preserve pluripotentprogenitor cells, as well as totipotent progenitor cells. In a preferredcase, the protein can preserve hematopoietic progenitor cells, butprogenitor cells from other tissues can also be preserved, includingnerve, muscle, skin, gut, bone, kidney, liver, pancreas, or thymusprogenitor cells. The progenitor cells capable of preservation accordingto the invention may express the CD34 antigen. More preferably, theprogenitor cells express both CD34 and the FLK2/FLT3 receptor. Stillmore preferably, the progenitor cells express the FLK2/FLT3 receptor butdo not express CD34. The protein can also be used to preserve cells thathave been modified to express FLK2/FLT3 receptors on their surface.Thus, the invention provides a protein that has significant bindingaffinity for FLK2/FLT3 receptor on the cells, wherein binding of theprotein with the FLK2/FLT3 receptor mediates the inhibition ofdifferentiation of the cells.

The invention further provides a method for preserving progenitor cells,comprising contacting progenitor cells with a protein encoded by anisolated nucleic acid comprising a nucleotide sequence defined by SEQ IDNO:1, a homolog thereof, or a fragment thereof that encodes an aminoacid sequence TNNVLQVT, in an amount sufficient to preserve theprogenitor cells.

The method of the invention is useful for preserving progenitor cellsfrom other species, particularly mammalian species. The progenitor cellspreferably comprise cells of hematopoietic origin. The method can beused for preserving any human progenitor cells that express the CD34antigen and/or the FLK2/FLT3 receptor. Alternatively, the method can beused to preserve any murine progenitor cells that express the Scaantigen, but that do not express mature blood cell lineage antigens.

The method can comprise contacting the progenitor cells with the proteinin vitro, ex vivo, or in vivo. In addition, the method can furthercomprise contacting the progenitor cells with FLK2/FLT3 ligand in anamount sufficient to selectively expand the number of progenitor cellswithout inducing differentiation thereof.

In another embodiment, the invention is a method of treating a mammal inneed of hematopoietic therapy, comprising:

a) obtaining a tissue sample from the mammal, the tissue samplecomprising hematopoietic progenitor cells;

b) culturing the progenitor cells in the presence of a protein thatpreserves the progenitor cells, to provide cultured cells enriched inthe progenitor cells, wherein the protein is encoded by an isolatednucleic acid comprising a nucleotide sequence defined by SEQ ID NO:1, ahomolog thereof, or a fragment thereof that encodes an amino acidsequence TNNVLQVT;

c) subjecting the mammal to conditions sufficient to effectmyeloablation; and

d) administering the cultured cells to the mammal following themyeloablation to reconstitute the hematopoietic system of the mammal.

According to the method, the myeloablation conditions can comprise bonemarrow irradiation, whole body irradiation, or chemically-inducedmyeloablation.

In another embodiment, the invention is a method of enriching progenitorcells, comprising culturing progenitor cells in a progenitor-preservingamount of a protein encoded by an isolated nucleic acid comprising anucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or afragment thereof that encodes an amino acid sequence TNNVLQVT, whereinthe protein specifically preserves the progenitor cells, and wherein theculturing is performed under conditions permitting preservation ofprogenitor cells while permitting the number of differentiated cells todecrease.

The method can be used to enrich progenitor cells, such as primitiveprogenitor cells, as well as mature progenitor cells. Preferably, theprogenitor cells are at least substantially free of stromal cells.

The culturing conditions used in the method can include culturing in amedium containing a cytotoxic agent that exhibits selective toxicity forproliferating cells. Suitable cytotoxic agents include, for example,adriamycin, cyclophosphamide, taxol or other taxane, cisplatin, or5-fluorouracil.

In still another embodiment, the invention is a method of improvinghematopoietic competence in a mammal, comprising:

a) culturing a tissue sample comprising hematopoietic progenitor cellsin a growth medium containing a protein encoded by an isolated nucleicacid comprising a nucleotide sequence defined by SEQ ID NO:1, a homologthereof, or a fragment thereof that encodes an amino acid sequenceTNNVLQVT, in an amount sufficient to preserve the progenitor cells andto provide cultured cells enriched in the progenitor cells; and

b) transfusing the enriched cultured cells to the mammal to provideprogenitor cells for generating blood cellular components in the mammal.

According to the method, the tissue sample can comprise peripheralblood, umbilical cord blood, placental blood, or bone marrow.Preferably, the tissue sample is autologous to the mammal. It is alsopreferred that the tissue sample is at least substantially free ofstromal cells. The method can further comprise ablating hematopoietictissues in the mammal prior to the transfusing.

In yet a further embodiment, the invention is an improvement to a methodof transfecting an exogenous DNA sequence into somatic cells, in whichthe improvement comprises transfecting progenitor cells selectivelypreserved by a protein encoded by an isolated nucleic acid comprising anucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or afragment thereof that encodes an amino acid sequence TNNVLQVT.

In another embodiment, the invention is a composition for preservingviability of progenitor cells ex vivo, comprising a cell growth mediumand a protein that preserves progenitor cells, wherein the protein isencoded by an isolated nucleic acid comprising a nucleotide sequencedefined by SEQ ID NO:1, a homolog thereof, or a fragment thereof thatencodes an amino acid sequence TNNVLQVT.

In a still further embodiment, the invention is a method for preservingprogenitor cells in a mammal, comprising:

a) administering to the mammal a protein that specifically preservesprogenitor cells, the protein being encoded by an isolated nucleic acidcomprising a nucleotide sequence defined by SEQ ID NO:1, a homologthereof, or a fragment thereof that encodes an amino acid sequenceTNNVLQVT, in an amount sufficient to preserve progenitor cells of themammal in a substantially non-proliferative state;

b) exposing the mammal to myeloablative conditions sufficient to effectablation of proliferating myeloid cells but sparing non-proliferatingprogenitor cells; and

c) following the exposing, reestablishing proliferation ordifferentiation of the preserved progenitor cells.

According to the method, the reestablishing can comprise administeringto the mammal a cytokine in an amount sufficient to improve theviability of the progenitor cells. The viability-improving cytokine canbe IL-1, IL-3, IL-6, IL-11, KL, or a combination thereof. The method canbe further modified such that the reestablishing comprises administeringto the mammal a proliferation-stimulating amount of the FLK2/FLT3ligand.

These and other advantages of the present invention will be appreciatedfrom the detailed description and examples that are set forth herein.The detailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention have been chosen for purposes ofillustration and description, but are not intended in any way torestrict the scope of the invention. The preferred embodiments ofcertain aspects of the invention are shown in the accompanying drawings,wherein:

FIG. 1 is a map of a cloning vector pCR2.1-DLA manufactured by ligatinga cDNA according to the invention in the EcoRI site of the cloningvector pCR2.1.

FIG. 2 is a direct amino acid sequence comparison of the mannose lectindescribed by Gowda et al. (1994) and the derived amino acid sequence ofthe protein encoded by the nucleic acid of the invention.

FIG. 3 is a map of a cloning vector pCR2.1-DLA(D) manufactured byligating a mutated cDNA in the EcoRI site of the cloning vector pCR2.1.

FIG. 4 is a map of a cloning vector pBS-SpDLA manufactured by ligating arecombinant fragment in the EcoRI site of the cloning vector pBluescriptSK+.

FIG. 5 is a map of a cloning vector pCR2.1-SpM1(D) manufactured byligating a mutated recombinant clone in the EcoRI site of the cloningvector pCR2.1.

FIG. 6 is a map of a recombinant expression vector pBIN-VicPromanufactured by subcloning the vicilin promoter obtained from the pCW66vector in the EcoRI/ClaI site of the plant binary vector pBIN19 forAgrobacterium-mediated transformation.

FIG. 7 is a map of a recombinant expression vector pBINVicPro-SpDLAmanufactured by ligating a recombinant fragment in the EcoRI/SacI siteof the pBINVicPro vector.

FIG. 8 is a map of a recombinant expression vector pBINVicPro-SpDLA(D)manufactured by ligating a mutated recombinant clone in the EcoRI siteother pBINVicPro vector.

FIG. 9 is a map of a recombinant expression vector pGEX4T-1-DLAmanufactured by ligating a wild-type cDNA clone in the EcoRI/SalI siteof the E. coli expression vector pGEX4T-1.

FIG. 10 is a map of a recombinant expression vector pGEX4T-1-DLA(D)manufactured by ligating a mutant cDNA clone in the EcoRI/XhoI site ofthe E. coli expression vector pGEX4T-1:

FIG. 11 is an electrophoretogram of a Southern blot of total proteinextracts of E. coli cells transformed with the recombinant expressionvectors pGEX4T-1-DLA and pGEX4T-1-DLA(D).

FIG. 12 is an electrophoretogram of a Western blot of purifiedGST-fusion proteins with and without cleavage by thrombin.

FIG. 13A is a graph showing that a crude extract of an E. coli culturecontaining expressed FRIL specifically stimulates hFLK2/FLT3 3T3 cells;FIG. 13B is a graph showing that the same extract does not stimulateuntransfected 3T3 cells.

FIG. 14A is a histogram showing that purified recFRIL preserves cordblood mononuclear cells in a dose-responsive manner; FIG. 14B is ahistogram showing that purified recFRIL preserves hematopoieticprogenitors in a dose-responsive manner.

FIG. 15 is a histogram showing that the protein encoded by a nucleicacid of the invention is sufficient to preserve progenitor cells invitro, whereas a cytokine cocktail fails to preserve such cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to an isolated nucleic acid encoding aprotein that preserves progenitor cells, and, therefore, the inventionfurther includes a method of using the nucleic acid in producing theencoded protein, and a method of using the encoded protein in preservingprogenitor cells.

Applicants have isolated and sequenced a nucleic acid having thesequence defined by SEQ ID NO:1, and homologs thereof, includinghomologs in other species. The invention further comprises uniquefragments of the nucleic acid of SEQ ID NO:1 and its homologs.

The protein encoded by the nucleic acid sequence defined by SEQ ID NO:1has the amino acid sequence defined by SEQ ID NO:2. But the inventionalso encompasses isolated nucleic acid molecules that encode uniqueportions of the protein specified as SEQ ID NO:2. Specifically, theinvention includes nucleic acids that encode proteins that contain thesequence TNNVLQVT, which is a part of SEQ ID NO:2, as well as functionalequivalents thereof.

“Nucleic acid,” as used herein, means any deoxyribonucleic acid (DNA) orribonucleic acid (RNA) that encodes in its nucleotide sequence a proteinas described herein or a unique fragment thereof. The fragment can be anoligonucleotide (i.e., about 8 to about 50 nucleotides in length) or apolynucleotide (about 50 to about 2,000 or more nucleotides in length).For example, nucleic acids include messenger RNA (mRNA), complementaryDNA (cDNA), genomic DNA, synthetic DNA or RNA, and the like. The nucleicacid can be single stranded, or partially or completely double stranded(duplex). Duplex nucleic acids can be homoduplex or heteroduplex.

The invention specifically includes nucleic acids that have a nucleotidesequence including the sequence defined by SEQ ID NO:1, or a homologthereof, or unique fragments thereof. In the present specification, thesequence of a nucleic acid molecule that encodes the protein isconsidered homologous to a second nucleic acid molecule if thenucleotide sequence of the first nucleic acid molecule is at least about30% homologous, preferably at least about 50% homologous, and morepreferably at least about 65% homologous to the sequence of the secondnucleic acid molecule. In the case of nucleic acids having highhomology, the nucleotide sequence of the first nucleic acid molecule isat least about 75% homologous, preferably at least about 85% homologous,and more preferably at least about 95% homologous to the nucleotidesequence of the second nucleic acid molecule. For example, a test forhomology of two nucleic acid sequences is whether they hybridize undernormal hybridization conditions, preferably under stringenthybridization conditions.

Given the nucleic acid sequence disclosed herein, the artisan canfurther design nucleic acid structures having particular functions invarious types of applications. For example, the artisan can constructoligonucleotides or polynucleotides for use as primers in nucleic acidamplification procedures, such as the polymerase chain reaction (PCR),ligase chain reaction (LCR), Repair Chain Reaction (RCR), PCRoligonucleotide ligation assay (PCR-OLA), and the like. Oligonucleotidesuseful as probes in hybridization studies, such as in situhybridization, can be constructed. Numerous methods for labeling suchprobes with radioisotopes, fluorescent tags, enzymes, binding moieties(e.g., biotin), and the like are known, so that the probes of theinvention can be adapted for easy detectability.

Oligonucleotides can also be designed and manufactured for otherpurposes. For example, the invention enables the artisan to designantisense oligonucleotides, and triplex-forming oligonucleotides, andthe like, for use in the study of structure/function relationships.Homologous recombination can be implemented by adaptation of the nucleicacid of the invention for use as targeting means.

As a new and specific nucleotide sequence is disclosed herein, theartisan will recognize that the nucleic acid can be produced by anysynthetic or recombinant process such as is well known in the art.Nucleic acids according to the invention can further be modified toalter biophysical or biological properties by means of techniques knownin the art. For example, the nucleic acid can be modified to increaseits stability against nucleases (e.g., “end-capping”), or to modify itslipophilicity, solubility, or binding affinity to complementarysequences. Methods for modifying nucleic acids to achieve specificpurposes are disclosed in the art, for example, in Sambrook et al.(1989) and, the disclosure of which is incorporated by reference herein.Moreover, the nucleic acid can include one or more portions ofnucleotide sequence that are non-coding for the protein of interest.

The skilled artisan appreciates that, if an amino acid sequence (primarystructure) is known, a family of nucleic acids can then be constructed,each having a sequence that differs from the others by at least onenucleotide, but where each different nucleic acid still encodes the sameprotein. For example, if a protein has been sequenced but itscorresponding gene has not been identified, the gene can be acquiredthrough amplification of genomic DNA using a set of degenerate primersthat specify all possible sequences encoding the protein.

The protein encoded by the nucleic acid of the invention, and functionalanalogs of the encoded protein, are essentially pure. For the purposesof this specification, “essentially pure” means that the protein andfunctional analogs are free from all but trace amounts of other proteinsas well as of materials used during the purification process. A proteinis considered to be essentially pure if it is at least 85%, preferablyat least 90%, and more preferably at least 95% pure. Methods forpurifying proteins are known in the art.

Determination of whether two amino acid sequences are substantiallyhomologous is, for the purpose of the present specification, based onFASTA searches in accordance with Pearson et al. (1988). In the presentspecification, the amino acid sequence of a first protein is consideredto be homologous to that of a second protein if the amino acid sequenceof the first protein has at least about 20% amino acid sequenceidentity, preferably at least about 40% identity, and more preferably atleast about 60% identity, with the sequence of the second protein. Inthe case of proteins having high homology, the amino acid sequence ofthe first protein has at least about 75% sequence identity, preferablyat least about 85% identity, and more preferably at least about 95%identity, with the amino acid sequence of the second protein.

The protein encoded by the nucleic acid of the present invention furtherincludes functional homologs. A protein is considered a functionalhomolog of another protein for a specific function, as described below,if the homolog has the same function as the other protein. The homologcan be, for example, a fragment of the protein, or a substitutionaddition, or deletion mutant of the protein.

As is also known, it is possible to substitute amino acids in a sequencewith equivalent amino acids. Groups of amino acids known normally to beequivalent are:

(a) Ala(A), Ser(S), Thr(T), Pro(P), Gly(G);

(b) Asn(N), Asp(D), Glu(E), Gln(Q);

(c) His(H), Arg(R), Lys(K);

(d) Met(M), Leu(L), Ile(I), Val(V); and

(e) Phe(F), Tyr(Y), Trp(W).

Substitutions, additions, and/or deletions in the amino acid sequencescan be made as long as the protein encoded by the nucleic acid of theinvention continues to satisfy the functional criteria described herein.An amino acid sequence that is substantially the same as anothersequence, but that differs from the other sequence by means of one ormore substitutions, additions, and/or deletions, is considered to be anequivalent sequence. Preferably, less than 50%, more preferably lessthan 25%, and still more preferably less than 10%, of the number ofamino acid residues in a sequence are substituted for, added to, ordeleted from the protein encoded by the nucleic acid of the invention.

As used herein, “progenitor cell” refers to any normal somatic cell thathas the capacity to generate fully differentiated, functional progeny bydifferentiation and proliferation. Progenitor cells include progenitorsfrom any tissue or organ system, including, but not limited to, blood,nerve, muscle, skin, gut, bone, kidney, liver, pancreas, thymus, and thelike. Progenitor cells are distinguished from “differentiated cells,”the latter being defined as those cells that may or may not have thecapacity to proliferate, i.e., self-replicate, but that are unable toundergo further differentiation to a different cell type under normalphysiological conditions. Moreover, progenitor cells are furtherdistinguished from abnormal cells such as cancer cells, especiallyleukemia cells, which proliferate (self-replicate) but which generallydo not further differentiate, despite appearing to be immature orundifferentiated.

Progenitor cells include all the cells in a lineage of differentiationand proliferation prior to the most differentiated or the fully maturecell. Thus, for example, progenitors include the skin progenitor in themature individual. The skin progenitor is capable of differentiation toonly one type of cell, but is itself not fully mature or fullydifferentiated.

Production of some mature, functional blood cells results fromproliferation and differentiation of “unipotential progenitors,” i.e.,those progenitors that have the capacity to make only one type of bloodcell. For red blood cell (erythrocyte) production, a unipotentialprogenitor called a “CFU-E” (colony forming unit-erythroid) has thecapacity to generate two to 32 mature progeny cells.

Various other hematopoietic progenitors have been characterized. Forexample, hematopoietic progenitor cells include those cells that arecapable of successive cycles of differentiating and proliferating toyield up to eight different mature hematopoietic cell lineages. At themost primitive or undifferentiated end of the hematopoietic spectrum,hematopoietic progenitor cells include the hematopoietic “stem cells.”These rare cells, which represent from about 1 in 10,000 to about 1 in100,000 of the cells in the bone marrow, and the most primitive cellshave the capacity to generate >10¹³ mature blood cells of all lineagesand are responsible for sustaining blood cell production over the lifeof an animal. They reside in the marrow primarily in a quiescent state,and may form identical daughter cells through a process called“self-renewal.” Accordingly, such uncommitted progenitor cells can bedescribed as being “totipotent,” i.e., both necessary and sufficient forgenerating all types of mature blood cells. Progenitor cells that retaina capacity to generate all blood cell lineages but that can notself-renew are termed “pluripotent.” Cells that can produce some but notall blood lineages and can not self-renew are termed “multipotent.”

The protein encoded by the nucleic acid of the invention is useful topreserve any of these progenitor cells, including unipotent progenitorcells, pluripotent progenitor cells, multipotent progenitor cells,and/or totipotent progenitor cells. The protein is useful in thepreservation and maintenance of progenitor cells in hematopoietictissues as well as in non-hematopoietic tissues, such as those mentionedabove.

The recombinant protein encoded by the nucleic acid of the invention isespecially useful in preserving hematopoietic progenitors in mammalssuch as humans, mice, rats, etc. In the human, hematopoietic progenitorcells can be identified as belonging to a class of cells defined bytheir expression of a cell surface antigen designated CD34. These cellsmay be referred to as “CD34⁺” cells. In the mouse, hematopoieticprogenitor cells may be referred to as “Sca⁺Lin⁻” cells, reflectingtheir cell surface antigen signature. Other mammalian species exhibitsimilar signature properties identifying hematopoietic progenitor cells.Hematopoietic progenitors can also be identified by their expression ofthe FLK2/FLT3 receptor.

Human hematopoietic progenitor cells that express the CD34 antigenand/or the FLK2/FLT3 receptor are referred to herein as “primitiveprogenitor cells.” Therefore, primitive progenitor cells includeCD34⁺FLK2/FLT3⁻ cells, CD34⁻FLK2/FLT3⁺ cells, and CD34⁺FLK2/FLT3⁺ cells.By contrast, hematopoietic cells that do not express either the CD34antigen or the FLK2/FLT3 receptor (i.e., CD34⁻FLK2/FLT3⁻ cells) arereferred to as “mature progenitor cells.”

Preferably, the recombinant protein is effective to preserve progenitorcells that express the CD34 antigen and/or the FLK2/FLT3 receptor. Theprogenitor cells can include cells modified to express the CD34 antigenor FLK2/FLT3 receptors on their surface. In a preferred case, theprotein has significant binding affinity for FLK2/FLT3 receptor on thecells, wherein binding of the protein with the FLK2/FLT3 receptormediates the inhibition of differentiation of the cells. The proteinencoded by the nucleic acid of the invention has been designated“FLK2/FLT3 Receptor-Interacting Lectin,” abbreviated “FRIL,” to describethis phenomenon, but this designator is used for convenience and shouldnot be understood to definitionally ascribe any specific property to, orany origin of, the protein.

The recombinant protein mediates “preservation” of progenitor cells. Bythis is meant that the protein inhibits differentiation of theprogenitor cells without depleting the progenitor cell population. Insome cases, the inhibition of differentiation is accompanied byproliferation of the progenitor cell population. In other cases, theinhibition of differentiation is induced without proliferation of theprogenitor cell population. In particular, by inhibiting differentiationprocesses, it is meant that the peptide significantly lowers the rate atwhich cells differentiate, and it may in fact completely stop theseprocesses. While the mechanism by which the protein acts is not itselfunderstood, one theoretical possibility is that the protein maintainsprogenitor cells in a quiescent or G0 state of the cell cycle.Regardless of the actual mechanism of its action, however, the proteindoes preserve progenitor cells without killing the cells in significantnumbers. In this sense, the recombinant protein is significantlydistinguished from factors that inhibit or interfere with cellularprocesses (e.g., DNA replication, protein synthesis), and that therebyinduce significant cell mortality.

As a result of the present invention, numerous utilities becometechnically feasible. The method of the invention can include contactingthe progenitor cells with the recombinant protein in vitro, ex vivo, orin vivo. “In vitro” methods include methods such as laboratoryexperimental methods which are performed wholly outside a living body.While cells can be acquired from a living organism for use in vitro, itis understood that the cells will not be returned to the body. In vitromethods are commonly employed in experimental settings to advanceunderstanding of particular systems. “Ex vivo” methods include clinicalmethods in which cells are manipulated outside the body of an organism,e.g., a patient, with the specific purpose of reimplanting some cellsback into the organism to achieve a desired therapeutic purpose. “Invivo” methods are performed within the body of the organism, withoutrequiring explantation or tissue sampling and manipulation.

For example, the recombinant protein finds a utility, inter alia, inthat it enables ex vivo preservation of hematopoietic progenitor cellsisolated from either normal or malignant (e.g., leukemic) bone marrow.Accordingly, the protein can be employed in the culture of mononuclearcells derived from a source of such cells, for example, from bonemarrow, umbilical cord blood, placental blood, or peripheral blood.Alternatively, the recombinant protein can be used in conjunction withgrowth factors such as colony stimulating factors (CSFs) (e.g., IL3,GM-CSF, G-CSF, M-CSF), interleukins (e.g., IL1 through IL18) and KL invitro to selectively induce proliferation and terminal differentiationof mature progenitors while preserving a significantly enrichedpopulation of primitive progenitors. U.S. Pat. Nos. 5,472,867 and5,186,931 describe representative methods of using CSFs and interleukins(ILs) to expand progenitor cell populations in the contexts of,respectively, cancer chemotherapy and bone marrow transplants. In apreferred case according to the present invention, the method canfurther includes contacting the progenitor cells with FLK2/FLT3 ligandin an amount sufficient to selectively expand the number of progenitorcells without inducing differentiation thereof.

The recombinant protein also enhances survival of progenitor cells whencultured in vitro. For example, a process of in vitro selection can beused that involves using the protein to preserve progenitor cells in asubstantially quiescent state in culture, while using a cytotoxic agentthat exhibits selective toxicity for proliferating cells, e.g., to killcells passing through cell cycle (“cycling cells”). Suitable cytotoxicagents include, for example, compounds such as adriamycin,cyclophosphamide, taxol or other taxane, cisplatin, 5-fluorouracil, andthe like. The method is useful to preserve quiescent progenitor cells.The method is effective even when the progenitor cells are substantiallyfree of stromal cells, which are considered to normally be necessary forprogenitor cell maintenance and proper hematopoietic reconstitution. Therecombinant protein improves the ability to functionally select stemcells either alone or with other factors. Such functional selectionmethods, include the method reported by Berardi et al. (1995) whereselection is made using a combination of KL and IL3 with 5-FU.

By preserving progenitor cells in a quiescent state, the protein encodedby the nucleic acid of the invention preserves normal progenitor cells,while the cycling cells are killed. For example, ex vivo purgingprotocols can be used to selectively eliminate neoplastic cells bytargeting the elimination of actively cycling cells. Once theprogenitors cells have been purged of malignant cycling cells, they canbe returned to the patient, and permitted to resume normal proliferationand differentiation. In one especially useful scenario, the recombinantprotein allows for functional selection of normal progenitor cells froma leukemic bone marrow.

Such functional selection and purification of primitive stem cells canalso be used to enable allogeneic transplant procedures. In situationswhere autologous tissue is not available for transplant, culturingallogeneic cells in the presence of the protein encoded by the nucleicacid of the invention will result in the selection of stem cells anddepletion of T lymphocytes and other effector cells. This will enabletransplant of progenitors while inhibiting a graft versus host reaction.Such stem cells acquire within the recipient immunological tolerance ofthe recipient's histocompatibility antigens.

It is a further advantage of the invention that it enables preservationof cells for periods and under conditions that permit shipment of cells,e.g., by mail, to distant locations for transplantation.

The recombinant protein also enables ex vivo manipulation ofhematopoietic progenitor cells for use in gene therapy by preservingcells in liquid culture. For example, by preserving hematopoieticprogenitor cells in culture for more than two weeks, the protein enablesincreased targeting efficiency by viral vectors that enternon-replicating cells (e.g., vectors such as adeno-associated viruses),and provides longer periods for the evaluation of the resultant cellpopulations to determine efficiency of transfection. Thus, in anotherembodiment, the method can be used in conjunction with methods oftransfecting an exogenous DNA sequence into somatic cells. The methodcan then include transfecting progenitor cells selectively preserved bythe recombinant protein.

The invention also has utility in conjunction with therapies, e.g.,cancer therapies, which employ irradiation. Specifically, because therecombinant protein preserves progenitor cells in a quiescent state,administration of the recombinant protein to a mammalian subject in vivoallows the use of increased levels of total body irradiation toeliminate neoplastic cells, while leaving quiescent cells relativelyunaffected. The protein can be employed in conjunction with othercytoprotective substances such as IL-1 to obtain an enhanced orcomplementary effect.

Thus, the method can involve treating a mammalian subject in need ofhematopoietic therapy. In particular, the recombinant protein can beused to improve hematopoietic competence in a mammal, i.e., the mammal'sability to generate functional mature blood elements. For example, atissue sample including hematopoietic progenitor cells can be obtainedfrom the subject. Then the tissue sample can be cultured ex vivo in agrowth medium containing the recombinant protein to preserve theprogenitors, while allowing cycling cells to proliferate, differentiateand die. The cultured cells become significantly enriched in theprimitive progenitor cells. Meanwhile, the mammal can be subjected toconditions sufficient to effect myeloablation, e.g., bone marrowirradiation, whole body irradiation, or chemically-inducedmyeloablation. Finally, the progenitor-enriched cultured cells can beadministered or transfused to the mammal following the myeloablation togenerate blood cellular components in the mammal, thereby reconstitutingthe hematopoietic system of the mammal. The method can use a tissuesample comprising peripheral blood, umbilical cord blood, placentalblood, or bone marrow. Preferably, the tissue sample is autologous tothe mammal. The tissue sample can also be at least substantially free ofstromal cells.

While described here as an autologous procedure, the skilledpractitioner will recognize that the methods can be readily adapted totransplant of progenitor-enriched cells from one individual to another.Again, when autologous tissue is not available for transplant, culturingallogeneic cells in the presence of the encoded protein can be used toinduce selection of stem cells and depletion of T lymphocytes and othereffector cells. The transplanted progenitor cells acquire within therecipient immunological tolerance of the recipient's histocompatibilityantigens, thereby mitigating graft vs. host reactions.

The invention further includes a composition for preserving viability ofprogenitor cells ex vivo or in vitro. The composition comprises aculture medium suitable for growth and maintenance of mammalian cells inculture, along with an amount of the recombinant protein sufficient topreserve progenitor cells as described herein. Virtually any cell ortissue culture medium can be modified for the preservation ofprogenitors in this way. Suitable standardized culture media are known,including, for example, Minimum Essential Medium Eagle's (MEM),Dulbecco's Modified Eagle's Medium (DMEM), McCoy's 5A Modified Medium,Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, RPMI-1640,specialized variant formulations of these media, and the like. Suchmedia can be supplemented using sera (e.g., fetal bovine serum) buffers(e.g., HEPES), hormones, cytokines, growth factors, or other desiredcomponents. Numerous media are available commercially, e.g., from SigmaChemical Co., St. Louis, Mo.

Ready-to-use receptacles, e.g., blood bags, media bags and bottles,microtiter plates, tissue and cell culture flasks, roller bottles, shakeflasks, culture dishes, and the like, can also be provided with theprotein encoded by the nucleic acid of the invention (with or withoutculture medium or other active components) to promote storage and/orculture of progenitor cells. The protein allows the artisan to storeprogenitor cells under refrigeration, at ambient temperature, or in anincubator at 37° C. The ability of the protein to preserve cells atambient temperatures is particularly useful for transporting cells.

Also, the invention includes a method for preserving progenitor cells ina mammal in vivo. In this approach, the method comprises administeringto the mammal the recombinant protein in an amount sufficient topreserve progenitor cells of the mammal in a substantiallynon-proliferative state. The mammal is then exposed to myeloablativeconditions sufficient to effect ablation of proliferating myeloid cellsbut sparing non-proliferating progenitor cells. Following the ablativetreatment, proliferation or differentiation of the preserved progenitorcells is reestablished, usually by administering to the mammal acytokine in an amount sufficient to improve the viability of theprogenitor cells. Preferred viability-improving cytokines include, forexample, FLK2/FLT3 ligand, IL1, IL3, IL6, IL11, KL, or a combinationthereof. According to this method, the recombinant protein can be usedto enhance autologous bone marrow transplantation techniques in whichlethal doses of radiation and/or chemotherapy are followed by reinfusionof stored marrow.

An effective amount of recombinant protein can be administered to amammal by any convenient approach, such as parenteral routes, e.g.,intravenous injection, or enteral routes, e.g., oral administration.Oral administration routes are expected to be useful since naturalsource lectins typically resist oral/gastric degradation, and canexhibit substantial bioavailability by this approach (Pusztai et al.1995). The skilled artisan recognizes the utility and limitations ofvarious methods of administration and can adjust dosage accordingly.

Other therapeutic utilities also present themselves to the skilledpractitioner as being enabled by the invention. Such other utilitiesinclude, for example, expanding progenitor cell populations ex vivo toincrease chances of engrafation, improving conditions for transportingand storing progenitor cells, and removing a fundamental barrier toenable gene therapy to treat and cure a broad range of life-limitinghematologic diseases such as sickle cell anemia and thalassemia.

The protein encoded by the nucleic acid of the invention can also beused as a specific probe for the identification or localization ofprogenitor cells. Since the protein binds specifically to primitiveprogenitor cells, a composition including the protein linked to adetectable moiety such as a fluorescent marker can be used tospecifically label and identify progenitor cells. Thus, cell sorting toisolate progenitor cells can be performed, as can histologiclocalization of progenitor cells in tissues, and other methods known inthe art. The skilled artisan can select the type or marker moiety to beemployed and the method of isolating cells according to the task to beperformed, since numerous methods of labeling proteins are known in theart.

The protein encoded by the nucleic acid of the invention can be used toisolate FLK2/FLT3-R-expressing progenitor cells. The protein ispreferably linked to a chemical group or to an object to assist in theisolation. For example, the protein can be chemically linked to magneticbeads. The multivalent nature of the protein is particularly useful forisolation of cells, such as primitive hematopoietic progenitors, whichexpress low levels of FLK2/FLT3-R (i.e., less than about 5,000receptors/cell). Similar methods using antibodies linked to magneticbeads require significantly higher levels of cell surface receptors.

Making and Using the Nucleic Acid of the Invention

The nucleic acid sequence of the invention can be isolated from anatural source, such as being derived from legume plants. Legumes suchas the garden pea or the common bean are plants (“leguminous plants”)from a family (Leguminosae) of dicotyledonous herbs, shrubs, and treesbearing (nitrogen-fixing bacteria) nodules on their roots. These plantsare commonly associated with their seeds (e.g., such as the garden peaor the common bean, etc.). More specifically, the nucleic acid can beisolated from members of the tribe Phaseoleae. In particular, thenucleic acid can be obtained from Dolichos lab lab, known, as hyacinthbeans and other common names throughout the world, from varieties of thecommon bean (Phaseolus vulgaris), e.g., red kidney beans, white kidneybeans, etc., and from Vigna sinensis, commonly known as the black-eyedpea. In its native form isolated from such natural sources, the nucleicacid appears to encode a mannose/glucose-specific legume lectin. Anexemplary isolation of the nucleic acid of the invention from Dolichoslab lab is described hereinbelow.

The entire gene or additional fragments of the gene are preferablyisolated by using the known DNA sequence or a fragment thereof as aprobe. To do so, restriction fragments from a genomic or cDNA libraryare identified by Southern hybridization using labeled oligonucleotideprobes derived from SEQ ID NO:1.

DNA according to the invention can also be chemically synthesized bymethods known in the art. For example, the DNA can be synthesizedchemically from the four nucleotides in whole or in part by methodsknown in the art. Such methods include those described in Caruthers(1985). DNA can also be synthesized by preparing overlappingdouble-stranded oligonucleotides, filling in the gaps, and ligating theends together. See, generally, Sambrook et al. (1989) and Glover et al.(1995).

DNA expressing functional homologs of the protein can be prepared fromwild-type DNA by site-directed mutagenesis. See, for example, Zoller etal. (1982); Zoller (1983); and Zoller (1984); McPherson (1991).

The DNA obtained can be amplified by methods known in the art. Onesuitable method is the polymerase chain reaction (PCR) method describedin Saiki et al. (1988), Mullis et al., U.S. Pat. No. 4,683,195, andSambrook et al. (1989). It is convenient to amplify the clones in thelambda-gt10 or lambda-gt11 vectors using lambda-gt10- orlambda-gt11-specific oligomers as the amplimers (available fromClontech, Palo Alto, Calif.).

Larger synthetic nucleic acid structures can also be manufactured havingspecific and recognizable utilities according to the invention. Forexample, vectors (e.g., recombinant expression vectors) are known whichpermit the incorporation of nucleic acids of interest for cloning andtransformation of other cells. Thus, the invention further includesvectors (e.g., plasmids, phages, cosmids, etc.) which incorporate thenucleotide sequence of the invention, especially vectors which includethe gene for expression of the protein encoded by the nucleic acid ofthe invention.

The DNA of the invention can be replicated and used to expressrecombinant protein following insertion into a wide variety of hostcells in a wide variety of cloning and expression vectors. The host canbe prokaryotic or eukaryotic. The DNA can be obtained from naturalsources and, optionally, modified. The genes can also be synthesized inwhole or in part.

Cloning vectors can comprise segments of chromosomal, non-chromosomaland synthetic DNA sequences. Some suitable prokaryotic cloning vectorsinclude plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC,pKSM and RP4. Prokaryotic vectors also include derivatives of phage DNAsuch as M13fd, and other filamentous single-stranded DNA phages.

Vectors for expressing proteins in bacteria, especially E. coli, arealso known. Such vectors include the pK233 (or any of the tac family ofplasmids), T7, and lambda. P_(L). Examples of vectors that expressfusion proteins are PATH vectors described, in Dieckmann and Tzagoloff(1985). These vectors contain DNA sequences that encode anthranilatesynthetase (TrpE) followed by a polylinker at the carboxy terminus.Other expression vector systems are based on beta-calactosidase (pEX);maltose binding protein (pMAL); glutathione S-transferase (pGST). See.,e.g., Smith (1988) and Abath (1990).

Vectors useful for cloning and expression in yeast are available. Asuitable example is the 2μ circle plasmid.

Suitable cloning/expression vectors for use in mammalian cells are alsoknown. Such vectors include well-known derivatives of SV40, adenovirus,cytomegalovirus (CMV) retrovirus-derived DNA sequences. Any suchvectors, when coupled with vectors derived from a combination ofplasmids and phage DNA, i.e., shuttle vectors, allow for the isolationand identification of protein coding sequences in prokaryotes.

Further eukaryotic expression vectors are known in the art (e.g.,Southern et al. (1982); Subramani et al. (1981); Kaufmann et al. (1982);Kaufmann et al. (1982); Scahill et al. (1983); Urlaub et al. (1980).

The expression vectors useful in the present invention contain at leastone expression control sequence that is operatively linked to the DNAsequence or fragment to be expressed. The control sequence is insertedin the vector in order to control and to regulate the expression of thecloned DNA sequence. Examples of useful expression control sequences arethe lac system, the trp system, the tac system, the trc system, majoroperator and promoter regions of phage lambda, the control region of fdcoat protein, the glycolytic promoters of yeast, e.g., the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,e.g., Pho5, the promoters of the yeast alpha-mating factors, andpromoters derived from polyoma, adenovirus, retrovirus, and simianvirus, e.g., the early and late promoters or SV40, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells and their viruses or combinations thereof.

Useful expression hosts include well-known prokaryotic and eukaryoticcells. Some suitable prokaryotic hosts include, for example, E. coli,such as E. coli SG-936, E. coli HB101, E. coli W3110, E. coli X1776, E.coli X2282, E. coli DHI, and E. coli MRCl, Pseudomonas, Bacillus, suchas B. subtilis, and Streptomyces. Suitable eukaryotic cells includeyeasts and other fungi, insect, animal cells, such as COS cells and CHOcells, human cells and plant cells in tissue culture.

Fusion Proteins

The protein can be expressed in the form of a fusion protein with anappropriate fusion partner. The fusion partner preferably facilitatespurification and identification. Increased yields can be achieved whenthe fusion partner is expressed naturally in the host cell. Some usefulfusion partners include beta-galactosidase (Gray et al. 1982); trpE(Itakura et al. 1977); protein A (Uhlen et al. 1983); glutathioneS-transferase (Johnson 1989; Van Etten et al. 1989); and maltose bindingprotein (Guan et al. 1987; Maina et al. 1988; Riggs 1990).

Such fusion proteins can be purified by affinity chromatography usingreagents that bind to the fusion partner. The reagent can be a specificligand of the fusion partner or an antibody, preferably a monoclonalantibody. For example, fusion proteins containing beta-galactosidase canbe purified by affinity chromatography using an anti-beta-galactosidaseantibody column (Ullman 1984). Similarly, fusion proteins containingmaltose binding protein can be purified by affinity chromatography usinga column containing cross-linked amylose; see Guan, European PatentApplication 286,239, incorporated herein by reference.

Optionally, the DNA that encodes the fusion protein is engineered sothat the fusion protein contains a cleavable site between the proteinand the fusion partner. The protein can occur at the amino-terminal orthe carboxy-terminal side of the cleavage site. Both chemical andenzymatic cleavable sites are known in the art. Suitable examples ofsites that are cleavable enzymatically include sites that arespecifically recognized and cleaved by collagenase (Keil et al. 1975);enterokinase (Hopp et al. 1988); factor Xa (Nagai et al. 1987); andthrombin (Eaton et al. 1986). Collagenase cleaves between proline and Xin the sequence Pro-X-Gly-Pro wherein X is a neutral amino acid.Enterokinase cleaves after lysine in the sequence Asp-Asp-Asp-Asp-Lys.Factor Xa cleaves after arginine in the sequence Ile-Glu-Gly-Arg.Thrombin cleaves between arginine and glycine in the sequenceArg-Gly-Ser-Pro.

Specific chemical cleavage agents are also known. For example, cyanogenbromide cleaves at methionine residues in proteins.

The recombinant protein is purified by methods known in the art. Suchmethods include affinity chromatography using specific antibodies.Alternatively, the recombinant protein can be purified using acombination of ion-exchange, size-exclusion, and hydrophobic interactionchromatography using methods known in the art. These and other suitablemethods are described, e.g., in Marston (1987).

Mixtures of proteins can be separated by, for example, SDS-PAGE inaccordance with the method of Laemmli (1970). The molecular weights weredetermined by resolving single bands on SDS-PAGE and comparing theirpositions to those of known standards. The method is understood by thosein the art to be accurate within a range of 3-5%. Molecular weights canvary slightly between determinations.

Fragments and Probes

As noted, the invention also includes fragments of the nucleic acidspecified as SEQ ID NO:1. Such fragments include primers and probeswhich are useful as tools in numerous molecular engineering techniques.The fragment can be used as a primer (“amplimer”) to selectively amplifynucleic acid, such as genomic DNA, total RNA, etc. The fragment can alsobe an oligonucleotide complementary to a target nucleic acid molecule,i.e., the fragment can be a probe. In either case, the oligonucleotidecan be RNA or DNA. The length of the oligonucleotide is not critical, aslong as it is capable of hybridizing to the target molecule. Theoligonucleotide should contain at least 6 nucleotides, preferably atleast 10 nucleotides, and, more preferably, at least 15 nucleotides.There is no upper limit to the length of the oligonucleotide. Longerfragments are more difficult to prepare and require longer hybridizationtimes. Therefore, the oligonucleotide should not be longer thannecessary. Normally, the oligonucleotide will not contain more than 50nucleotides, preferably not more than 40 nucleotides, and, morepreferably, not more than 30 nucleotides.

Methods for determining whether a probe nucleic acid molecule recognizesa specific target nucleic acid molecule in a sample are known in theart. Generally, a labeled probe that is complementary to a nucleic acidsequence suspected of being in a sample is prepared. Preferably, thetarget nucleic acid molecule is immobilized. The presence of probehybridized to the target nucleic acid molecule indicates the presence ofthe nucleic acid molecule in the sample. Examples of suitable assaymethods are described in Dallas et al. (1975); Grunstein et al. (1975);U.S. Pat. No. 4,731,325, U.S. Pat. No. 4,683,195, U.S. Pat. No.4,882,269, and PCT publication WO 90/01069, all of which areincorporated herein by reference.

The probes described above are labeled in accordance with methods knownin the art. The label can be a radioactive atom, an enzyme, or achromophoric moiety.

Methods for labeling oligonucleotide probes have been described, forexample, in Leary et al. (1983); Renz et al. (1984); Richardson et al.(1983); Smith et al. (1985); and Meinkoth et al. (1984).

The label can be radioactive. Some examples of useful radioactive labelsinclude ³²P, ¹²⁵I, ¹³¹I and ³H. Use of radioactive labels have beendescribed in U.K. patent document 2,034,323, and U.S. Pat. Nos.4,358,535, and 4,302,204, each incorporated herein by reference.

Some examples of non-radioactive labels include enzymes, chromophores,atoms and molecules detectable by electron microscopy, and metal ionsdetectable by their magnetic properties.

Some useful enzymatic labels include enzymes that cause a detectablechange in a substrate. Some useful enzymes and their substrates include,for example, horseradish peroxidase (pyrogallol ando-phenylenediaimine), beta-galactosidase (fluoresceinbeta-D-galactopyranoside), and alkaline phosphatase(5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The useof enzymatic labels have been described in U.K. 2,019,404, and EP63,879, each incorporated herein by reference, and by Rotman (1961).

Useful chromophores include, for example, fluorescent, chemiluminescent,and bioluminescent molecules, as well as dyes. Some specificchromophores useful in the present invention include, for example,fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone,luminol.

The labels can be conjugated to the antibody or nucleotide probe bymethods that are well known in the art. The labels can be directlyattached through a functional group on the probe. The probe eithercontains or can be caused to contain such a functional group. Someexamples of suitable functional groups include, for example, amino,carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.

Alternatively, labels such as enzymes and chromophoric molecules can beconjugated to the antibodies or nucleotides by means of coupling agents,such as dialdehydes, carbodiimides, dimaleimides, and the like.

The label can also be conjugated to the probe by means of a ligandattached to the probe by a method described above and a receptor forthat ligand attached to the label. Any of the known ligand-receptorcombinations is suitable. Some suitable ligand-receptor pairs include,for example, biotin-avidin or -streptavidin, and antibody-antigen. Thebiotin-avidin combination is preferred.

In any case, methods for making and using nucleic acid probes are welldocumented in the art. For example, see Keller et al. (1993) and Hameset al. (1995).

The following examples are provided to assist in a further understandingof the invention. The particular materials and conditions employed areintended to be further illustrative of the invention and are notlimiting upon the reasonable scope thereof.

EXAMPLE 1 RNA Isolation and cDNA Synthesis

Total RNA was prepared from mid-maturation Dolichos lab lab seeds storedat −70° C. following the procedure of Pawloski et al. (1994). Poly(A⁺)RNA was obtained from this total RNA using the PolyATract mRNA IsolationSystem (Promega) according to the manufacturer's instructions. Avianmyeloblastosis virus reverse transcriptase (Promega) was used togenerate cDNA from 0.5 μg poly(A⁺)RNA, or from 3.0 μg of total RNA,using 1 μg of oligo(dT) in standard reaction conditions (Sambrook et al.1989).

Polymerase Chain Reaction and cDNA Cloning

EXAMPLE 2A

Based on the amino acid sequence published by Gowda et al. (1994), twodegenerate oligonucleotide primers were designed using Phaseolus codonusage (Devereux et al. 1984): MLA AA (AG) TT (TC) GA (TC) CC (AT) AA(SEQ ID NO:3) (TC) CA (AG) GA (AG) GA MLZ TT (AT) CC (AG) TT (TC) TGCCA(AG) (SEQ ID NO:4) TCCCA

A 500+ bp product was amplified from cDNA prepared as described inExample 1, by 30 cycles of polymerase chain reaction (PCR), each cyclecomprising 40 s at 94° C., 40 s at 50° C., 60 s at 72° C., followed byan extension step at 72° C. for 10 min. Reactions were performed in 50μL containing 30 pmol of each primer, 0.2 mM deoxyribonucleotides and0.5 unit of AmpliTaq polymerase (Perkin Elmer) in the correspondingbuffer.

The 500 bp product obtained by PCR was cloned in the cloning vector,pCR2.1 (Invitrogen), and sequenced by sequenase dideoxy chaintermination (United States Biochemicals) using the following primers:GTACCGAGCTCGGAT (SEQ ID NO:5) TCTAGATGCATGCTCGAG (SEQ ID NO:6)This sequence was designated “FRILa,” as relating to the gene encodingthe protein of interest, designated “FRIL” as noted above.

EXAMPLE 2B

Based on the sequence of the FRILa amplified product, a specific primerwas prepared: MLX GTTGGACGTCAATTCCGATGTG (SEQ ID NO:7)

A degenerate primer corresponding to the first five amino acids of thesequence published by Gowda et al. (1994) was also prepared: MLI GC (TC)CA (AG) TC (TC) CT (TC) TC (SEQ ID NO:8) (TC) TT

The MLX and MLI primers were used in combination to amplify a 480 bpproduct from cDNA prepared as in Example 1, through 30 PCR cycles usingthe same conditions described above. This secondary amplified fragmentwas cloned in the pCR2.1 vector and sequenced as described above, andwas designated “FRILb.”

EXAMPLE 2C

The 3′ end of FRIL was obtained through rapid amplification of cDNA endsby polymerase chain reaction (RACE-PCR) (see, e.g., Frohman 1990) usingthe 5′/3′ RACEKIT (Boehringer Mannheim) according to the manufacturer'sinstructions. In the cDNA synthesis for the 3′ RACE, an oligo(dT) anchorprimer (“AP”) supplied with the kit was used, at a concentration of 32.5μM, using the standard conditions described above in Example 1. APGACCACGCGTATCGATGTCGAC (SEQ ID NO:9)

Nested PCR amplifications were performed using the AP anchor primer incombination with a specific primer having the following sequence: MLBAAGTTAGACAGTGCAGGAAAC (SEQ ID NO:10)The amplification conditions were again 30 cycles of 40 s at 94° C., 40s at 55° C., 60 s at 72° C. each, with an extension step at 72° C. for10 min. A 900+ bp product was obtained, which was subcloned in pCR2.1and sequenced as described above, and was designated “FRILc” (SEQ IDNO:1).

EXAMPLE 2D

To obtain the full length cDNA clone, the anchor primer AP was used incombination with a specific primer corresponding to the first 5 aminoacids encoded at the 5′-terminus: MLII GCACAGTCATTGTCATTTAG (SEQ IDNO:11)The full length cDNA was obtained through 30 cycles of PCR, each cyclecomprising 60 s at 94° C., 60 s at 58° C., 90 s at 72° C., with anextension step at 72° C. for 10 min. The reaction was performed in 100μL containing 30 pmol of each primer, 0.2 mM deoxyribonucleotide, 1.0unit of Pfu polymerase (Stratagene). The MLII and AP primers weredesigned to generate an EcoRI site at each end (3′ and 5′) of thepolynucleotide sequence. The full length cDNA was ligated into the EcoRIsite of the cloning vector pCR2.1, resulting in the final product“pCR2.1-DLA” illustrated schematically in FIG. 1.

EXAMPLE 3 The Nucleotide Sequence of FRIL

The FRILc clone obtained as described in Example 2C was sequencedcompletely using the dideoxy chain termination method. The nucleotidesequence of the full-length cDNA is: (SEQ ID NO:1) 1 GCACAGTCATTGTCATTTAG TTTCACCAAG TTTGATCCTA ACCAAGAGGA 51 TCTTATCTTC CAAGGTCATGCCACTTCTAC AAACAATGTC TTACAAGTCA 101 CCAAGTTAGA CAGTGCAGGA AACCCTGTGAGTTCTAGTGC GGGAAGAGTG 151 TTATATTCTG CACCATTGCG CCTTTGGGAA GACTCTGCGGTATTGACAAG 201 CTTTGACACC ATTATCAACT TTGAAATCTC AACACCTTAC ACTTCTCGTA251 TAGCTGATGG CTTGGCCTTC TTCATTGCAC CACCTGACTC TGTCATCAGT 301TATCATGGTG GTTTTCTTGG ACTCTTTCCC AACGCAAACA CTCTCAACAA 351 CTCTTCCACCTCTGAAAACC AAACCACCAC TAAGGCTGCA TCAAGCAACG 401 TTGTTGCTGT TGAATTTGACACCTATCTTA ATCCCGATTA TGGTGATCCA 451 AACTACATAC ACATCGGAAT TGACGTCAACTCTATTAGAT CCAAGGTAAC 501 TGCTAAGTGG GACTGGCAAA ATGGGAAAAT AGCCACTGCACACATTAGCT 551 ATAACTCTGT CTCTAAAAGA CTATCTGTTA CTAGTTATTA TGCTGGGAGT601 AAACCTGCGA CTCTCTCCTA TGATATTGAG TTACATACAG TGCTTCCTGA 651ATGGGTCAGA GTAGGGTTAT CTGCTTCAAC TGGACAAGAT AAAGAAAGAA 701 ATACCGTTCACTCATGGTCT TTCACTTCAA GCTTGTGGAC CAATGTGGCG 751 AAGAAGGAGA ATGAAAACAAGTATATTACA AGAGGCGTTC TGTGATGATA 801 TATGTGTATC AATGATTTTC TATGTTATAAGCATGTAATG TGCGATGAGT 851 CAATAATCAC AAGTACAGTG TAGTACTTGT ATGTTGTTTGTGTAAGAGTC 901 AGTTTGCTTT TAATAATAAC AAGTGCAGTT AGTACTTGT

The FRIL nucleotide sequence enabled inference of a derived amino acidsequence for the FRIL protein: (SEQ ID NO:2) AQSLSFSFTK FDPNQEDLIFQGHATSTNNV LQVTKLDSAG NPVSSSAGRV LYSAPLRLWE DSAVLTSFDT IINFEISTPYTSRIADGLAF FIAPPDSVIS YHGGFLGLFP NANTLNNSST SENQTTTKAA SSNVVAVEFDTYLNPDYGDP NYIHIGIDVN SIRSKVTAKW DWQNGKIATA HISYNSVSKR LSVTSYYAGSKPATLSYDIE LHTVLPEWVR VGLSASTGQD KERNTVHSWS FTSSLWTNVA KKENENKYIT RGVL

A comparative illustration of the derived FRIL amino acid sequence withthe reported amino acid sequence of the mannose lectin as determined byGowda et al. (1994) is shown in FIG. 2. The single sequence derived forFRIL protein comprises domains that correspond directly and withsubstantial homology to the α subunit (SEQ ID NO:12) and β subunit (SEQID NO:13) of the protein described by Gowda et al. (1994). When the βsubunit of the Gowda et al. protein is assigned to the N-terminal domainand is followed linearly by the α subunit, the arrangement of thepolypeptides shows homology to other legume lectins. However, thederived FRIL amino acid sequence comprises an insert of seven amino acidresidues (aa27-34) that does not occur in the protein described by Gowdaet al. Several other differences between the amino acid sequences of thetwo proteins are also readily discernible from FIG. 2.

EXAMPLE 4 Site-Specific Mutagenesis

To establish functionality of homologs of the protein encoded by theFRIL cDNA, a mutation was made in the FRIL cDNA clone. The domains ofthe derived protein and the pea lectin that include the mutation siteare shown below: FRIL . Y L N P D Y G . D P N Y I H I G I D V (SEQ IDNO:14) Pea F Y . N A A W D P S N R D R H I G I D V (SEQ ID NO:15)It is known that the asparagine residue (the highlighted “N”) in the pealectin is involved in binding to its saccharide ligand. Thecorresponding asparagine in FRIL (position 141 of the amino acidsequence, based on the sequence including the 15 amino acid signalpeptide) was mutated to aspartic acid (“D”). This mutation wasdesignated “N141D” for convenience.

To introduce the mutation recombinant PCR was performed (Higuchi 1990).Two PCR reactions were carried out separately on the full length cDNAusing two primers that contain the same mutation and produce twoproducts with an overlapping region: MutI CCATAATCGGGATCAAGATAGGTG (SEQID NO:16) MutII CACCTATCTTGATCCCGATTATGG (SEQ ID NO:17)

The primary PCR products were purified with the QIAquick PCRPurification kit (QIAGEN), according to the manufacturer's instructions.The overlapping primary products were then combined and amplifiedtogether in a single second reaction using flanking primers: M1 ForwAACTCAGCCGCACAGTCATTGTCA (SEQ ID NO:18) APEcoRIGAATTCGACCACGCGTATCGATGTCGAC (SEQ ID NO:19)

Both the primary and the secondary PCR reactions were performed in 100μL containing 50 pmol of each primer, 0.4 mM deoxyribonucleotide and 1.0unit Pfu polymerase (Stratagene) in the corresponding buffer. Theprimary PCR reaction amplified the two separate fragments in 30 cycles,each cycle comprising 40 s at 94° C., 40 s at 50° C., 72° C. 60 s, withan extension step at 72° C. for 10 min. The second PCR reactionamplified the recombinant fragment in 12 cycles using the sameconditions reported above.

The resulting full-length fragment contained the mutation. Therecombinant mutated product was cloned in the EcoRI site of the cloningvector pCR2.1, as illustrated schematically in FIG. 3, and sequenced asdescribed above. This plasmid is referred to as “pCR2.1-DLA(D).”

EXAMPLE 5 Construction of Plant Expression Vectors and Nicotiana tabacumTransformation

Recombinant PCR was used to modify the 5′ ends of both the wild-type andthe mutant FRIL clones, to introduce a signal peptide for entry of theprotein into the endoplasmic reticulum. Following the procedure ofHiguchi (1990), the sequence encoding the signal peptide and thefull-length cDNA clones were amplified in two separate primary PCRreactions. The signal peptide sequence was obtained from theamplification of the binary vector pTA4, harboring the complete sequenceof the α-amylase inhibitor gene (Hoffnan et al. 1982; Moreno et al.1989).

The following primers were used for amplification of the signal peptidesequence: Sigforw GAATTCATGGCTTCCTCCAAC (SEQ ID NO:20) SigrevTGACTGTGCGGCTGAGTTTGCGTGGGTG (SEQ ID NO:21)

The primers M1Forw (SEQ ID NO:18) and APEcoRI (SEQ ID NO:19) used foramplification of the FRIL cDNA in Example 4 above, were again used toamplify the FRIL cDNA.

The primers used for the secondary reactions were Sigforw and APEcoRI,which were designed to generate EcoRI sites at the 5′ and the 3′ ends.Both the primary and the secondary PCR reactions were performed asdiscussed above for the site-directed mutagenesis.

The wild-type recombinant product SpDLA was cloned in the EcoRI site ofthe pBluescript SK+ cloning vector (Stratagene) to give the vectorpBS-SpDLA, as shown in FIG. 4. The mutant SpDLA(D) was cloned in thesame site of the cloning vector pCR2.1 to give the vector pCR2.1-SpM1,as shown in FIG. 5. The nucleotide sequence of each PCR product wasdetermined as described above to verify the correct attachment of thesignal peptide. The nucleotide sequence of SpDLA is defined by SEQ IDNO:22, and the derived amino acid sequence is defined by SEQ ID NO:23.

EXAMPLE 6

A binary vector was constructed for seed-specific expression of FRIL.For seed expression, the vicilin promoter obtained from the pCW66(Higgins et al. 1988) was cloned in EcoRI/KpnI sites of the plantexpression vector pBIN19, to form pBINVicPro, as illustrated in FIG. 6.Downstream of the vicilin promoter, the SpDLA cDNA sequence was ligatedinto the EcoRI/SacI site giving rise to the pBINVicPro-SpDLA, which isillustrated in FIG. 7. The mutated cDNA clone SpDLA(D) was ligated inEcoRI site of the pBINVicPro vector to yield pBINVicPro-SpDLA(D), whichis illustrated in FIG. 8. No additional termination sequences wereadded, relying instead on the stop codons and the polyadenylation siteof the DLA and DLA(D) cDNA clones. Both vectors were transferred intoAgrobacterium tumefaciens strain LBA4404 according to the freeze-thawprocedure reported by An et al. (1988).

Agrobacterium-mediated transformation of Nicotiana tabacum leaf diskswas carried out and assayed as described (Horsch et al. 1985) usingLBA4404 harboring the seed-specific expression vector pBINVicPro-SpDLA(FIG. 9). Kanamycin-resistant plants (resistance being conferred bytransformation with the pBIN19-based vectors that carry the gene) werescored for their ability to form roots in two consecutive steps ofpropagation in Murashige-Skoog medium containing 3% of sucrose andkanamycin sulfate (Sigma) 100 mg/mL.

EXAMPLE 7 Expression of Recombinant FRIL in E. coli

The FRIL wild-type cDNA and mutant clones (without signal peptides),were Ligated into the EcoRI/SalI and EcoRI/XhoI of the expression vectorpGEX 4T-1 (Pharmacia), to form the expression constructs pGEX-M1 andpGEX M1(D), respectively illustrated in FIGS. 9 and 10. The host E. colistrain, BL21(D3), was purchased from Novagen, and transformed with theabove construct using the calcium chloride method (see Sambrook et al.1989; Gelvin et al. 1988; Altabella et al. 1990; and Pueyo et al. 1995).The induction of the tac promoter (Ptac) was achieved by adding IPTG(isopropyl-β-D-thiogalactopyranoside) (Sigma) at a 1.0 mM finalconcentration when the cells reached an optical density of 0.4-0.6 at600 nm. The cultures were allowed to grow for 12 h at 37° C. after theaddition of IPTG. Control non-induced cultures were maintained undersimilar conditions. The cells were lysed by treatment with 4 mg/mLlysozyme in phosphate-buffered saline containing 1% TRITON® X-100.

Total cellular protein was extracted from transformed E. coli cells andanalyzed on SDS-PAGE on a 15% gel using a standard procedure (Sambrooket al. 1989). The cells from 1 mL of E. coli culture were suspended inthe same volume of loading buffer (50 mM Tris HCl pH 6.8, 100 mM DTT, 2%SDS, 10% glycerol, 0.1% bromophenol blue) and vortexed. Followingtransfer to a nitrocellulose membrane, protein was stained withCoomassie Brilliant Blue R250. A representative separation is shown inFIG. 11, with the lanes identified in Table 1, below. TABLE 1 Key toFIG. 11 Lane No. Content 1 Molecular Mass Marker (Bio-Rad) 2 TotalProtein Extract from Non-Induced BL21(D3) pGEX-M1 3 Total ProteinExtract from Induced BL21(D3) pGEX-M1 4 Total Protein Extract fromNon-Induced BL21(D3) pGEX-M1(D) 5 Total Protein Extract from InducedBL21(D3) pGEX-M1(D)

The separation of proteins in FIG. 11 shows that the induced cells((lanes 3, 5) both produced an abundant polypeptide having a molecularmass of about 60 kDa (indicated by arrow). The non-induced cells failedto produce any significant amount of this protein (lanes 2, 4).

EXAMPLE 8 Purification of Recombinant FRIL

Induced E. coli cells (200 mL) as described in Example 7 were harvestedafter 12 h induction at 37° C. by centrifugation at 5000 g for 10 min.The pellet was washed with 50 mM Tris-HCl pH 8.0, 2 nM EDTA, andresuspended in 1/10 vol of 1% TRITON surfactant in TBS (20 mM Tris pH7.5, 500 mM NaCl). The cells were lysed by adding 4 mg/mL of lysozymeand incubating at room temperature for 30-66 min. After centrifugationat 5000 g, the supernatant containing the total soluble proteins wasdiscarded and the resulting pellet, comprising the inclusion bodies andcontaining the accumulated the recombinant fusion protein, was extractedwith 8 M guanidine-HCl (Martson et al. 1993).

The recombinant fusion protein solubilized by guanidine-HCl was purifiedon GST-Sepharose beads (Pharmacia) according the manufacturer'sinstructions and eluted in 1 mL of reduced glutathione (Sigma). Samplesof the purified fusion proteins were cleaved with thrombin (Novagen)using 5 cleavage units/mL purified fusion protein.

For immunoblot analysis (western blot), the purified proteins wereseparated by SDS-PAGE in general accordance with the method described inExample 7. The gel was equilibrated in transfer buffer (25 mM Tris pH8.3, 192 mM Glycine, 20% MeOH) and blotted onto nitrocellulose (Bio-Rad)for 1 h at 100 V using a Bio-Rad electrotransfer apparatus. Non-specificbinding was blocked by incubating the blots for at least 1 h in 1×TBS(20 mM Tris pH 7.5, 500 mM NaCl) containing 3% gelatin. Blotting wasfollowed by incubation with a primary antibody (a polyclonal rabbitserum raised against the N-terminal peptide of the β-subunit of thePhaseolus vulgaris homolog of FRIL, 1:100 dilution, 3 h), followed byincubation with a secondary antibody (goat anti-rabbit IgG conjugated tohorseradish peroxidase at 1:1000 dilution for 1 h). The blots werewashed and the color developed with the color development reagent(Bio-Rad). A representative result is shown in FIG. 12, with the lanesidentified in Table 2, below TABLE 2 Key to FIG. 12 Lane No. Content 1Purified Fusion Protein M1 2 Purified Fusion Protein M1(D) 3 Control 4Purified Fusion Protein M1 After Cleavage with Thrombin 5 PurifiedFusion Protein M1(D) After Cleavage with Thrombin 6 Control

The separation shown in FIG. 12 demonstrates that the two forms offusion protein have similar molecular masses of about 60 kDa, and thatthrombin cleaved both types of fusion protein to produce a newpolypeptide of molecular mass 30 kDa.

EXAMPLE 9 Recombinant FRIL Specifically Stimulates Proliferation of 3T3Cells Expressing the FLK2/FLT3 Receptor

The recombinant protein interacts with the mammalian FLK2/FLT3 tyrosinekinase receptor. A specific and quantitative biological assay using NIH3T3 fibroblasts transfected either with a chimeric receptor having theextracellular portion of the murine FLK2/FLT3 receptor combined with theintracellular portion of the human Fms receptor (Dosil et al. 1993) orwith the full length human receptor (Small et al. 1994) can be used toevaluate lectin biological activity during purification. Serial two-folddilutions of lectin samples across rows of a 96 well plate allowed forgreater than a thousand-fold range to access FLK2/FLT3 3T3 biologicalactivity. Either the murine or human FLK2/FLT3 ligand (FL) (Lyman et al.1993; Hannum et al. 1994) or the recombinant protein encoded by thenucleic acid of the invention rescues FLK2/FLT3-transfected cells fromdeath in this assay.

Specifically, 3T3 cells cultured in tissue culture plates (BectonDickinson Labware, Lincoln Park, N.J.) are removed from the plates bywashing cells twice in Hank's buffered saline solution (HBSS; GibcoLaboratories, Grand Island, N.Y.). Non-enzymatic cell dissociationbuffer (Gibco) is added for 15 minutes at room temperature. Theresulting cells are washed in medium. FLK2/FLT3 3T3 cells are culturedat a final concentration of 3,000 cells per well in a volume of 100 μLof serum-defined medium containing 10 mg/mL rhIL1-α 10% AMV (Gibco) and90% Dulbecco's modification of Eagle's medium (DMEM; Gibco) in 96 wellplates. Under these assay conditions, cells die after two to four daysof culture in a humidified incubator at 37° C. and 5% CO₂ unlessexogenously added ligand rescues cells from death. Each 96 well platecontains calf serum, which stimulates all 3T3 cells, as a positivecontrol and medium only as a negative control (“blank”). Full-lengthFms-transfected 3T3 cells (biological response shown in Tessler et al.1994) serve as receptor-transfected control target cells, and parent 3T3cells serve as untransfected control cells. Proliferation and cellsurvival is quantitated by addition of XTT (Diagnostic Chemicals Ltd,Charlottetown, Prince Edward Island, Canada), which is a tetraformazansalt cleaved by actively respiring cells (Roehm et al. 1999),quantitated spectrophotometrically using a Vmax kinetic plate reader(Molecular Devices Corp., Mountain View, Calif.), and recorded as eitherrelative activity (units/mL) or as specific activity (units/mg). Oneunit of biological activity is defined as the reciprocal dilution atwhich half-maximal stimulation of cells is detected.

The crude protein extract from the E. coli cultures described in Example7, above, was tested to determine whether expressed recFRIL possessedany capacity to stimulate FLK2/FLT3 3T3 cells using this assay. The datafrom this experiment are summarized in FIGS. 13A and 13B. Specifically,FIG. 13A is a graph showing that the crude extract of the E. coliculture containing expressed FRIL specifically stimulates hFLK2/FLT3cells; FIG. 13B is a graph showing that the same extract does notstimulate untransfected 3T3 cells. In FIGS. 13A and 13B, medium controlis represented by a solid line. The ordinate (absorbance) indicates cellviability measured by XTT at three days; the abscissa shows thereciprocal dilution of the extract sample. The apparent inhibition ofproliferation observed at higher concentrations (FIG. 13A) is notunderstood, but may relate to toxic components in the crude E. coliextract or the consequences of dose-related preservation of the 3T3fibroblasts.

EXAMPLE 10 Recombinant FRIL Preserves Mononuclear Cells and Progenitorsin Liquid Culture

The recFRIL protein preserves functional progenitors for at least twoweeks in liquid culture. FIGS. 14A and 14B illustrate the results of anexperiment in which recFRIL is shown to act in a dose-responsive mannerto preserve human cord blood progenitors.

FIGS. 14A and 14B show that recombinant FRIL preserves cord bloodmononuclear cells and progenitors in a dose-responsive manner in liquidculture. Cord blood mononuclear cells obtained by FICOLL-PAQUE®separation (Pharmacia Biotech, Piscataway, N.J.) were cultured inserum-free medium (X-VIVO 10, Biowittaker, Walkersville, Md.) at aconcentration of 200,000 cells/mL in a volume of 4 mL for two weekswithout medium changes. Harvested cells were pelleted and resuspended inX-VIVO 10 before determining viable cell number by trypan blue (GIBCO,Grand Island, N.Y.) exclusion. These results are shown in FIG. 14A. Theprogenitor number and capacity of harvested cells were assessed byplating the cells in complete serum-free, methylcellulose colony assaymedium (StemCell Technologies, Vancouver, BC, Canada). After two weeks,the resultant colonies were scored and the results are shown in FIG.14B. In FIGS. 14A and 14B, “blast” refers to colonies consisting ofprimitive, morphologically undifferentiated cells; “mix” refers tocolonies consisting of myeloid and erythroid cells; “erythroid” refersto colonies consisting of erythroid cells; and “myeloid” refers tocolonies consisting of myeloid cells. Cell number is shown on theordinate; the abscissa shows the reciprocal dilution of the sample.

To assess whether recFRIL acts directly or indirectly through accessorycells to preserve progenitor cells, cord blood mononuclear cells werefirst enriched for progenitors expressing the CD34 antigen byimmunomagnetic bead isolation (Dynal Corp., Lake Success, N.Y.). Fivehundred CD34⁺ cells were placed into wells containing 100 μL ofserum-free medium (BIT9500, StemCell Technologies) either in thepresence of recFL (PeproTech, Princeton, N.J.) or a cytokine cocktail ofrhIL3+rhIL6+rhIL11+rhTpo+FL (BioSource International, Camarillo, Calif.)in 96-well plates and cultured for four weeks without medium changes.The numbers of functional progenitors from these cultures were assessedby plating cells in complete serum-free methylcellulose colony assaymedium (StemCell Technologies). After two weeks, the resultant colonieswere scored and the results are shown in FIG. 15 (solid bars=recFRIL;open bars=cytokine cocktail). Clearly, progenitors were preserved onlythe recFRIL-containing cultures. Thus, purified recFRIL acts directly onprimitive hematopoietic progenitors.

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention those skilled in theart will realize that other and further embodiments can be made withoutdeparting from the spirit of the invention, and it is intended toinclude all such further modifications and changes as come within thetrue scope of the claims set forth herein.

BIBLIOGRAPHY

The following documents have been mentioned in the foregoingspecification, and are incorporated herein by reference for all thatthey disclose:

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1. An isolated nucleic acid molecule comprising a sequence that encodesa protein comprising an amino acid sequence as defined by SEQ ID NO:2, ahomolog thereof, or a fragment thereof that contains the sequenceTNNVLQVT.
 2. A isolated nucleic acid molecule according to claim 1,comprising a nucleotide sequence as defined by SEQ ID NO:1, a homologthereof, or a unique fragment thereof.
 3. An isolated nucleic acidmolecule that encodes a protein that preserves progenitor cells, whereinthe nucleic acid is isolated from a legume.
 4. An isolated nucleic acidaccording to claim 3, wherein the encoded protein comprises an aminoacid sequence as defined by SEQ ID NO:2 or a fragment thereof thatcontains the sequence TNNVLQVT.
 5. An isolated nucleic acid according toclaim 3, wherein the nucleic acid encodes a mannose/glucose-specificlegume lectin.
 6. An isolated nucleic acid according to claim 3, whereinthe nucleic acid is isolated from a legume from the tribe Phaseoleae. 7.An isolated nucleic acid according to claim 7, wherein the nucleic acidis isolated from red kidney beans, white kidney beans, hyacinth beans,or black-eyed peas.
 8. An isolated nucleic acid according to claim 3,wherein the nucleic acid comprises a nucleotide sequence as defined bySEQ ID NO:1 or a unique fragment thereof.
 9. An isolated nucleic acidaccording to claim 3, wherein the encoded protein preserves progenitorcells that are at least unipotent progenitor cells.
 10. An isolatednucleic acid according to claim 3, wherein the encoded protein preservesprogenitor cells that are pluripotent progenitor cells.
 11. An isolatednucleic acid according to claim 3, wherein the encoded protein preservesprogenitor cells that are totipotent progenitor cells.
 12. An isolatednucleic acid according to claim 3, wherein the encoded protein preservesprogenitor cells that are hematopoietic progenitor cells.
 13. Anisolated nucleic acid according to claim 3, wherein the encoded proteinpreserves progenitor cells that comprise nerve, muscle, skin, gut, bone,kidney, liver, pancreas, or thymus progenitor cells.
 14. An isolatednucleic acid according to claim 9, wherein the encoded protein preservesprogenitor cells that express the CD34 antigen.
 15. An isolated nucleicacid according to claim 9, wherein the encoded protein preservesprogenitor cells that express the FLK2/FLT3 receptor.
 16. An isolatednucleic acid according to claim 15, wherein the encoded proteinpreserves progenitor cells that are cells modified to express FLK2/FLT3receptors on their surface.
 17. An isolated nucleic acid according toclaim 16, wherein the encoded protein has significant binding affinityfor FLK2/FLT3 receptor on the cells, wherein binding of the encodedprotein with the FLK2/FLT3 receptor mediates the inhibition ofdifferentiation of the cells.
 18. A method for preserving progenitorcells, comprising contacting progenitor cells with a protein encoded byan isolated nucleic acid comprising a nucleotide sequence defined by SEQID NO:1, a homolog thereof, or a fragment thereof that encodes an aminoacid sequence TNNVLQVT, in an amount sufficient to preserve theprogenitor cells.
 19. A method according to claim 18, wherein theprogenitor cells comprise hematopoietic progenitor cells.
 20. A methodaccording to claim 19, wherein the progenitor cells comprise human cellsthat express the CD34 antigen and/or the FLK2/FLT3 receptor.
 21. Amethod according to claim 19, wherein the progenitor cells comprisemurine cells that express the Sca antigen but that do not express matureblood cell lineage antigens.
 22. A method according to claim 18, whereinthe method comprises contacting the progenitor cells with the protein invitro, ex vivo, or in vivo.
 23. A method according to claim 18, whereinthe method further comprises contacting the progenitor cells withFLK2/FLT3 ligand in an amount sufficient to selectively expand thenumber of progenitor cells without inducing differentiation thereof. 24.A method of treating a mammal in need of hematopoietic therapy,comprising: a) obtaining a tissue sample from the mammal, the tissuesample comprising hematopoietic progenitor cells; b) culturing theprogenitor cells in the presence of a protein that preserves theprogenitor cells, to provide cultured cells enriched in the progenitorcells, wherein the protein is encoded by an isolated nucleic acidcomprising a nucleotide sequence defined by SEQ ID NO:1, a homologthereof, or a fragment thereof that encodes an amino acid sequenceTNNVLQVT; c) subjecting the mammal to conditions sufficient to effectmyeloablation; and d) administering the cultured cells to the mammalfollowing the myeloablation to reconstitute the hematopoietic system ofthe mammal.
 25. A method according to claim 24, wherein themyeloablation conditions comprise bone marrow irradiation, whole bodyirradiation, or chemically-induced myeloablation.
 26. A method ofenriching progenitor cells, comprising culturing progenitor cells in aprogenitor-preserving amount of a protein encoded by an isolated nucleicacid comprising a nucleotide sequence defined by SEQ ID NO:1, a homologthereof, or a fragment thereof that encodes an amino acid sequenceTNNVLQVT, wherein the protein specifically preserves the progenitorcells, and wherein the culturing is performed under conditionspermitting preservation of progenitor cells while permitting the numberof differentiated cells to decrease.
 27. A method according to claim 26,wherein the progenitor cells include primitive progenitor cells.
 28. Amethod according to claim 26, wherein the progenitor cells includemature progenitor cells.
 29. A method according to claim 26, wherein theprogenitor cells are at least substantially free of stromal cells.
 30. Amethod according to claim 26, wherein the culturing conditions includeculturing in a medium containing a cytotoxic agent that exhibitsselective toxicity for proliferating cells.
 31. A method according toclaim 30, wherein the cytotoxic agent is adriamycin, cyclophosphamide,taxol or other taxane, cisplatin, or 5-fluorouracil.
 32. A method ofimproving hematopoietic competence in a mammal, comprising: a) culturinga tissue sample comprising hematopoietic progenitor cells in a growthmedium containing a protein encoded by an isolated nucleic acidcomprising a nucleotide sequence defined by SEQ ID NO:1, a homologthereof, or a fragment thereof that encodes an amino acid sequenceTNNVLQVT, in an amount sufficient to preserve the progenitor cells andto provide cultured cells enriched in the progenitor cells; and b)transfusing the enriched cultured cells to the mammal to provideprogenitor cells for generating blood cellular components in the mammal.33. A method according to claim 32, wherein the tissue sample comprisesperipheral blood, umbilical cord blood, placental blood, or bone marrow.34. A method according to claim 33, wherein the tissue sample isautologous to the mammal.
 35. A method according to claim 32, whereinthe tissue sample is at least substantially free of stromal cells.
 36. Amethod according to claim 32, further comprising ablating hematopoietictissues in the mammal prior to the transfusing.
 37. In a method oftransfecting an exogenous DNA sequence into somatic cells, theimprovement comprising transfecting progenitor cells selectivelypreserved by a protein encoded by an isolated nucleic acid comprising anucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or afragment thereof that encodes an amino acid sequence TNNVLQVT.
 38. Acomposition for preserving viability of progenitor cells ex vivo,comprising a cell growth medium and a protein that preserves progenitorcells, wherein the protein is encoded by a nucleic acid comprising anucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or afragment thereof that encodes an amino acid sequence TNNVLQVT.
 39. Amethod for preserving progenitor cells in a mammal, comprising: a)administering to the mammal a protein that specifically preservesprogenitor cells, the protein being encoded by an isolated nucleic acidcomprising a nucleotide sequence defined by SEQ ID NO:1, a homologthereof, or a fragment thereof that encodes an amino acid sequenceTNNVLQVT, in an amount sufficient to preserve progenitor cells of themammal in a substantially non-proliferative state; b) exposing themammal to myeloablative conditions sufficient to effect ablation ofproliferating myeloid cells but sparing non-proliferating progenitorcells; and c) following the exposing, reestablishing proliferation ordifferentiation of the preserved progenitor cells.
 40. A methodaccording to claim 39, wherein the reestablishing comprisesadministering to the mammal a cytokine in an amount sufficient toimprove the viability of the progenitor cells
 41. A method according toclaim 40, wherein the viability-improving cytokine is IL-1, IL-3, IL-6,IL-11, KL, or a combination thereof.
 42. A method according to claim 39,wherein the reestablishing comprises administering to the mammal aproliferation-stimulating amount of the FLK2/FLT3 ligand.